Evaluation of the sperm parameters, oxidative stress, and histopathological effects of vitamin B12 in preventing Helicobacter pylori-induced testicular toxicity: An experimental study

Abstract Background Helicobacter pylori (H. pylori) causes sterility by affecting the reproductive system. Vitamin B12 improves sperm quantity and function. Objective Vitamin B12 protection against H. pylori adverse effects was investigated. Materials and Methods 40 C57 male mice (6 wk) were randomly assigned to 4 equal groups (n = 10) including, group 1 (control without any intervention), group 2 (H), 3 (HP), and 4 (HB) received 1 × 109 colony forming unit (CFU) of H. pylori, 1 × 109 CFU of H. pylori+phosphate buffered saline, 1 × 109 CFU of H. pylori+50 μ g/kg vitamin B  12 intraperitoneally, respectively. In the induction groups, the H. pylori was orogasterically injected 3 times with 1 cc phosphate buffered saline throughout the day. Then testicular metrics, sperm motility, viability, quantity, and shape, plasma levels of malondialdehyde (MDA), superoxide dismutase, glutathione peroxidase, and total antioxidant capacity were measured. Also, testicular-tissue changes were examined using Johnson scores, tubular differentiation index, and spermatogenesis index. Vitamin B12, homocysteine, and testosterone serum levels were examined. Results The results showed a significantly lower Johnson score, tubular differentiation index, and spermatogenesis index, and serum level of testosterone and homocysteine as well as a higher MDA level in the H and HP groups than the HB group (p < 0.05). In contrast, the highest superoxide dismutase and glutathione peroxidase enzymes activity and total antioxidant capacity as well as the lowest serum level of MDA were found in the HB group compared to other groups (p < 0.05). Conclusion Vitamin B  12 increased antioxidant enzyme activity, enhanced sperm parameters, and decreased injury to testicular tissue. It can be used as a potent antioxidant in reducing testicular damage induced by H. pylori.


Introduction
Healthy reproduction is the foundation for the species' existence and perpetuation.
However, there has recently been an increase in accounts of decreasing male fertility (1).
According to estimates, infertility impacts millions of reproductive-aged partners globally (2).Male infertility accounts for approximately half of all infertility instances (3).Male infertility can be caused by a variety of factors, including hereditary abnormalities, steroid hormone disorders, hypogonadism, spermatogenesis failure, heavy alcohol consumption, ejaculation disorders, and reproductive infections.
A wide range of microorganisms, such as bacteria, viruses, and parasites, can invade the reproductive organs of men and initiate a cascade of inflammatory reactions that ultimately lead to a decrease in fertility of men (4).
Helicobacter pylori (H.pylori) is a microaerophilic, gram-negative bacterium with a spiral shape.It thrives in the stomach and triggers a robust cellular and immune system response, leading to chronic gastritis.In combination with bacterial factors, this response contributes to the development of peptic ulcers in 15-20% of infected people and gastric carcinoma in 1-3%, with variations based on the virulence of the infecting organisms and the geographical location (5).There is evidence to suggest that infection with H. pylori, particularly those that produce CagA, may have a detrimental impact on human reproductive capacity (6).The relationship between H. pylori infection and extra-gastrointestinal illnesses is often attributed to oxidative stress as a potential underlying mechanism.However, there are fewer studies on the effective role of antioxidant supplements in reducing oxidative stress caused by H. pylori infection.
Vitamin B 12 (- [5, 6-dimethylbenzimidazolyl] cobamidcyanide), also known as cobalamin due to the presence of cobalt in its chemical structure, is one of the 8 recognized B vitamins (7).Vitamin B 12 is essential for spermatogenesis (8).It   The first group was without any injection (control group).

Experiment and animals
The second group was induced with H. pylori after an overnight fast and orogasterically

H. pylori culture
The H. pylori strain ATCC43504 was cultivated in a microaerophilic environment at a temperature of 37°C on Helicobacter agar for a duration of 5-7 days.The colonies of H. pylori were selected randomly and then submerged in brucella broth that was supplemented with 5-10% fetal bovine serum.This suspension was incubated under microaerophilic circumstances at a temperature of 37°C for 24 hr (10).

Histopathological evaluation
The excised testes were immersed in a 10% formalin solution for 24 hr to facilitate fixation.
The paraffin slices underwent detachment and processing and were subsequently stained using hematoxylin and eosin staining.The testicles were assessed using tubular differentiation index (TDI), spermatogenesis index (SPI) (11), and modified Johnson scores (Table I) (12).To obtain the SPI, the ratio of active spermatogonia cells to inactive spermatogonia cells was calculated.TDI was obtained, and the percentage of spermatogenic seminiferous tubules or differentiated seminiferous tubules were recorded.100 seminiferous tubules were analyzed for the evaluation of Johnson score, TDI, and SPI (13).

Assessment of sperm parameters
The measurement of sperm concentration was conducted using a sperm counting chamber (Sperm Processor located, Aurangabad, India).A volume of 20 μl of the sample that was obtained was diluted with an equal volume of water.

Serum analyses
The levels of B12 (vitamin B 12 II, Roche,

Assessment of serum TAC
The TAC of serum was measured using the ferric-reducing antioxidant power method.The basis of this method is the ability of serum to Then, the optical absorption of the supernatant solution was recorded by a spectrophotometer (UNICO 2150-UV Spectrophotometer, China) at a wavelength of 523 nm (15).

Assessment of serum superoxide dismutase enzyme
To determine the amount of superoxide dismutase enzyme, the Zellbio (ZB-SOD96) kit was used according to the kit protocol.For this purpose, 10 × l of each serum was used and after adding the solutions, its absorbance was measured by a spectrophotometer (UNICO 2150-UV Spectrophotometer, China) at a wavelength of 420 nm.

Assessment of serum glutathione peroxidase enzyme
To determine the amount of glutathione peroxidase enzyme, both the kit of Zellbio (ZB-GPX96) and the hydrogen peroxide substrate in the kit were used.For this purpose, 10 µl of each serum was used and after adding the solutions, its absorbance was measured by a spectrophotometer (UNICO 2150-UV Spectrophotometer, China) at a wavelength of 412 nm.

Statistical analysis
All the studied data showed normal distribution.Collected data were expressed as the mean ± standard deviation (SD) and p < 0.05 was considered as significant.Where appropriate, ANOVA and independent-sample t test were used to define differences within groups.All the analysis was carried out in SPSS software version 22 (SPSS Science, Chicago, IL, USA).

Histopathology
The testicular tissues of the control group were structurally normal and contained an abundance of sperm cells with normal spermatogonia, layered in multiple layers on the seminiferous tubule (Figure 1A).In the control group, the tubular epithelium was A significant progress in the HB group was observed, where there was a partial recovery of germinal cells and spermatozoa (rounded arrow) with abundant spermatogenic cell layers (Figure 1D).Leydig cells and typical, weak sperm have been identified in the seminiferous tubules of the vitamin B 12 treatment group.

Comparison of histological examinations of mice testis
The testicular levels of Johnson scores, TDI, and SPI exhibited a significant decrease in both the H and HP groups when compared to the control and HB groups (p < 0.001).No statistically significant difference was observed between the control and the HB groups in terms of Johnson scores, TDI, and SPI (p = 0.09, Figure 2).

Sperm analysis
The average values of sperm concentration and progressive sperm motility were found to be considerably lower in the H and HP groups when compared to both the control group and the HB group (p < 0.001).There is no significant variation in the mean value of sperm normal morphology across the various groups (Figure 3).

Plasma factors
As shown in figure 4, the mean plasma levels of H. pylori, vitamin B 12 , testosterone, and homocysteine were significantly decreased in the H and HP groups compared to the control and HB groups (p < 0.001).In addition, no significant difference was observed between the control and HB groups with respect to plasma levels of H. pylori (p = 0.178), vitamin B 12 (p = 0.092), testosterone (p = 0.903), and homocysteine (p = 0.638).

The effect of H. pylori and vitamin B 12 on MDA level and TAC
In comparison to control group, the concentration of MDA was significantly higher in the H and HP groups (p < 0.001).In the HB group, the amount of MDA was significantly decreased compared to the HP and H groups (p < 0.001).Moreover, based on our findings, H. pylori infection decreased TAC relative to the control group (p < 0.001).When compared to the H and HP groups, vitamin B 12 administration in group 4 substantially averted the decrease in TAC (p < 0.001).Statistical analysis revealed no significant variation between the control and HB groups (Figure 5).

Discussion
Since H. pylori infection has been shown to be associated with the histological changes of the seminiferous tubules, the protective effect of vitamin B 12 against H. pylori-induced testicular damage was evaluated in the current study after 3 wk of treatment.The spermatogenesis is progressed nearly over the 4-6 wk in rat testes (16) (20).
Several studies have examined the protective effects of potent antioxidants (phytochemicals, vitamins, and enzymes) on the testicles of rodents (12,(21)(22).
In this study, vitamin B 12 was selected as it has previously shown to protect against testicular damage caused by cimetidine (23).Vitamin B 12 has also shown to play a part in the preservation of reproductive processes in the testis.Vitamin B 12 promotes DNA production, which aids in cell proliferation (24).Rodents deficient in vitamin B 12 have shown testicular pathological conditions characterized by notable diminishment in testis weight, shrinkage of the seminiferous tubules, and aplasia of spermatids and sperm (23,25).In oligospermically provoked male rats, for example, methylcobalamin at 1000 µg/kg (6 times a week for 5-10 wk) caused a significant rise in the width of the seminiferous tubules as well as sperm concentration (26).In addition, studies revealed that spermatogenesis would be enhanced when a high dose of vitamin B 12 is administered to patients with oligospermia in relation to fertility (27) (23).Our results in this study showed that there is a significant relationship between infection with H. pylori and the reduction of vitamin B 12 and the increase of serum homocysteine.
Increased ROS and decreased antioxidant defense lead to redox imbalance, decreased sperm motility, and DNA damage in sperm (30).
Due to the abundance of unsaturated fatty acids in their cell membranes, spermatozoa are extremely susceptible to the detrimental effects of ROS (31).
Oxidative stress is associated with an increase in the generation of oxygen-free radicals or an unbalance in the redox defense system.SOD, GPX, and catalase are the 3 major enzymatic components of this system in sperm (32).Our results showed that the activity of SOD and GPX was decreased in the H. pylori infected.As the quantity of ROS in the tissue rises, plasma membrane lipids become damaged and peroxidized, and internal components are destroyed, with MDA serving as a measure for lipid peroxidation (33).In our study, the oxidative stress and histopathological effects of vitamin B 12 pretreatment in the prevention of H. pylori injury in mice were evaluated, and it was discovered that vitamin B 12 supplementation significantly decreased plasma MDA levels and increased GPX and SOD activity.
Along with the results obtained from our research, it was reported that infection with H. pylori, especially the CagA strain, can be responsible for infertility in men through a destructive effect on sperm (24).
Furthermore, a study revealed that the presence of H. pylori infection leads to elevated levels of inflammatory cytokines in semen.These elevated levels have been associated with a decrease in sperm motility, potential sperm damage, and ultimately a decline in male fertility (7).
Finally, this study's findings reported that vitamin B 12 supplementation has a positive impact on preventing and treating complications of H. pylori infection and infertility.

Conclusion
The results of this study can help to explain that 40 healthy male C57 mice (6 wk old, weight 20-25 gr) were purchased from Pasture Institute, Iran.The mice were kept under controlled environmental conditions, with a consistent room temperature of 25 ± 2°C and a humidity range of 40-70%.The mice were housed in plastic cages containing hardwood chips as bedding material, as well as in polypropylene cages that provided a natural 12-hr light/dark cycle.The mice were provided with a standard laboratory pellet meal and were given unrestricted access to water.Mice were randomly divided into 4 groups (n = 10/each).

Subsequently, 10
μl of this diluted mixture was carefully placed into a sperm-counting chamber, allowing for the determination of the number of spermatozoa per milliliter.The assessment of sperm motility % was conducted using light microscopy at a magnification of ×40.To evaluate sperm movement, a 10 μl aliquot of the material was placed onto a slide that had been preheated, and the proportion of spermatozoa exhibiting progressive motion was documented.The evaluation of sperm morphology was conducted using the eosin/nigrosin staining method.Initially, 20 µl of washed spermatozoa in PBS was mixed with 40 µl of eosin (Merck, Darmstadt, Germany) for 5 min.Then, 60 µl of nigrosin (Merck, Darmstadt, Germany) was administered.Smears were produced for each sample, and 200 spermatozoa were counted using a CX31 OLYMPUS light microscope (magnification x100).The proportion of spermatozoa with head, neck, and tail abnormalities and total abnormalities were determined (13).
Switzerland), testosterone (Testosterone II, Roche, Switzerland), and H. pylori in the serum were assessed using the enzyme-linked immunosorbent assay kits (Elecsys 2010 and Cobas e411 analyzers) according to the manufacturer's instructions.The absorbance was measured spectrophotometrically at a wavelength of 450 nm.The determination of homocysteine serum levels was performed using high-performance liquid chromatography.

2 . 8 .
reduce ferric ions Fe +3 to Fe +2 in the presence of tripyridyltriazine (2, 4, 6-Tripyridyl-s-triazineTPTZ) (number 3682-35-7, Sigma, Germany).In this method, the reaction of Fe +2 with TPTZ reagent creates a blue-colored Fe +2 -TPTZ complex with a maximum light absorption property at a wavelength of 593 nm, which can be measured by increasing the concentration of the abovementioned complex by a spectrophotometer (UNICO 2150-UV Spectrophotometer, China) (14).Assessment of serum MDA To measure serum MDA, a working solution containing 0.5 gr of thiobarbituric acid (CAS-number 504-17-6, Merck, Germany) and 80 ml of 20% acetic acid (CAS-number 64-19-7, Merck, Germany) was used.The pH of this solution was adjusted to 3.5 using sodium hydroxide (NaOH) (CAS-number 1310-73-2, Merck, Germany) and its final volume was adjusted to 100 ml by adding 20% acetic acid.In the next step, 2.5 ml of this solution was poured into a glass test tube along with 100 µl of serum and 100 µl of sodium dodecyl sulfate (1.8%) (CAS-number 151-21-3, Merck, Germany).And the tubes were closed with an aluminum cap.The tubes were placed in a hot water bath for 1 hr and after cooling, they were centrifuged at 4000 rpm (Poland Mpw 260 r).
mostly unchanged and comprised Sertoli cells besides spermatocytes and spermatogonia on the basement membrane.Our result showed that infection with H. pylori resulted in testicular structural changes characterized by a reduced number of spermatozoa (Figures 1B and C, arrowhead).The seminiferous tubules in the H and HP groups had significant changes compared to the control group, such as seminiferous tubule deterioration and necrosis, reduced interstitial tissue with extensive intracellular spaces, spermatogonia cell loss, and reduction of mature sperm are all indicators of sperm dysfunction.
Superoxide dismutase (SOD) is one of the antioxidant enzymes responsible for preventing ROS production in cells.It converts anion superoxide (O 2 ) to low-risk hydrogen peroxide in cells and converts glutathione peroxidase (GPX) and catalase to water.In this study, infection with H. pylori significantly decreased SOD levels compared to the control group (p < 0.01).When compared to the H and HP groups, the results showed that vitamin B 12 had a positive effect on SOD and increased its activity.Compared to the control group, the specific activity of GPX was significantly (p < 0.01) reduced in the H and HP groups.Compared to corresponding controls, pre-treatment with vitamin B 12 restored this diminished activity (Figure 6).

Figure 1 .
Figure 1.Histopathological evaluations of adult mouse testicular tissue.A) Control, B) Group H, C) Group HP, and D) Group HB.In the control group, seminiferous tubules are arranged normally (black arrow).Abnormal arrangement and diminished seminiferous tubule diameter, vacuolation (white arrow), increased interstitial tissue (star), and decreased sperm density (arrowhead) in the lumens of the H and HP groups.In the vitamin B 12 + H. pylori groups, the seminiferous tubules were arranged normally and contained more sperm.Additionally, the seminiferous tubule walls were thicker (Magnifications: x400).

Figure 2 .
Figure 2. Comparison of testis of mice histological examinations within groups.Comparison of Johnson score, spermatogenesis index (SPI), and tubular differentiation index (TDI) within the control group, mice were induced with H. pylori (H group), mice were induced with H. pylori and PBS (HP group), and mice received H. pylori and vitamin B 12 .***Shows a significant difference at p = 0.000 between groups.

Figure 3 .
Figure 3. A) Sperm concentration, B) Sperm progressive motility, C) Sperm normal morphology between the control group, H (induced with H. pylori), HP (induced with H. pylori and PBS) and HB (induced with H. pylori and vitamin B 12 ) groups.The results are repeated 3 times and the Mean ± SD. ***Show a significant difference at p < 0.001 between groups.

Figure 4 .
Figure 4. Comparison of H. pylori, vitamin B 12 , testosterone hormone, and homocysteine between the control group with H (induced with H. pylori), HP (induced with H. pylori and PBS), and HB (induced with H. pylori and vitamin B 12 ) groups.The results are repeated 3 times and Mean ± SD. ***Shows a significant difference at p < 0.001 between groups.

Figure 6 .
Figure 6.The effect of vitamin B 12 supplementation on oxidative stress-related enzyme synthesis in infected mice with H. pylori.A) The influence of vitamin B 12 on SOD activity.B) The effects of vitamin B 12 on GPX activity.Values are presented as the Mean ± SD of 10 animals.**Significantly distinct from control (p < 0.01).H (H. pylori), HP (H.pylori and PBS), and HB (H.pylori and vitamin B 12 ) groups.
infection with H. pylori can lead to infertility by inducing harmful changes in testicular tissue and sperm parameters.In addition, H. pylori can cause harm to the reproductive system by destroying the antioxidant balance and decreasing the TAC, and activity of SOD and GPX enzymes.According to the results of the present investigation, vitamin B 12 is an effective antioxidant that can decrease oxidative stress, enhance sperm parameters, and reduce the rate of testicular tissue damage.The beneficial effects of vitamin B 12 on sperm parameters can be attributed to improve the efficacy of male reproductive organs, reduced homocysteine toxicity, and decreased ROS accumulation.However, additional research, predominantly clinical, is required to confirm these positive effects.
. The decreased Johnson score, TDI, and SPI in the H. pylori-infected group confirms the testicular toxicity of H. pylori infection.The data obtained from the serum level of SOD, GPX, MDA, and TAC showed that the testicular damage was associated with increased oxidative stress.While, the higher Johnson score, TDI, and SPI in the H.
(18)ri-infected + vitamin B 12 group than in the H.(18).For many years, research organizations have concentrated on the potential impact of H. pylori infections on sperm quality.H. pylori infection, particularly by isolates producing the CagA protein, Journal of Reproductive BioMedicine Volume 22, Issue no. 5. https://doi.org/10.18502/ijrm.v22i5.16439 . Our results showed that infection with Helicobacter leads to a decrease in serum levels of vitamin B 12 .International